Bacteria of the genus Yersinia are responsible for a variety of human diseases. Y. pestis causes the infamous disease Plague, which has regained prominence in public awareness due to its potential use as an agent of bioterrorism. In contrast, Y. pseudotuberculosis and Y. enterocolitica cause primarily gastrointestinal disease. However, despite the differences in disease symptoms, these three pathogenic Yersinia species are closely related, and share several common virulence determinants. Yersinia studies have provided fundamental insights into bacterial pathogenesis, including the first example of the widespread type three secretion system (T3SS). A critical component of all T3SSs is a specialized outer membrane pore-forming protein known as a secretin. However, secretin production can cause bacterial cell envelope stress. This is lethal to Y. enterocolitica unless a critical stress response known as the phage-shock-protein (Psp) system is functional. As a result, the Psp system of Y. enterocolitica is essential for its virulence. Our studies on the Psp system to date have identified its core components and begun to define their roles. We will base our future work on the hypotheses that regulation of the Y. enterocolitica Psp system is mediated by complex and dynamic protein- protein interactions, and that the activated system functions to counter problems associated with the cytoplasmic membrane, such as can be caused by a mislocalized secretin. To address these hypotheses we propose to: (1) Test various models of how the PspFABC proteins may constitute a signal transduction system that regulates psp gene expression via dynamic protein-protein interactions; (2) Analyze the regulatory and physiological functions of the PspB and PspC proteins, which play multiple essential roles in the system; (3) Directly analyze the connections between secretin toxicity, secretin mislocalization to the cytoplasmic membrane, and the function of the Psp system in Y. enterocolitica. These studies also have broad significance beyond Y. enterocolitica because secretin-containing systems critical for virulence, and the Psp system, are widespread in medically important bacteria. Therefore, by understanding the Psp system we will gain further insight into the essential ability of bacteria to respond to stressful conditions that occur during host infection.